Environmentally and endogenously-induced nucleic acid damage, and in particular deoxyribonucleic acid (“DNA”) damage, has long been associated with cancer, aging, neurological disorders, and heritable diseases. Measurements of DNA damage in human samples is therefore fundamentally valuable, both for delineating genotoxic environmental conditions that render cells vulnerable to mutations, and for revealing genetic factors that modulate susceptibility to DNA damage. For example, it is well established that heritable DNA repair deficiencies can promote cancer. (Vogelstein and Kinzler 2004.)
Although it is generally accepted that environmental conditions can induce DNA damage that is hazardous to human health, and that people deficient in the ability to repair DNA damage are more prone to diseases, measurement of DNA damage and repair in human samples is far from routine. Despite the multitude of industrial chemicals present in today's workplaces, environment, air, food, and water, fewer than 1000 such chemicals have been thoroughly characterized in terms of the risks that they pose to human health. This is at least because existing assays are labor-intensive, expensive, and technically challenging.
Many different approaches for analyzing nucleic acid damage and repair presently exist, but they each have their own distinct shortcomings. Many assays require the cells be homogenized in order to isolate and analyze DNA. This process makes it impossible to know which cell types are the most damaged unless cell types are separated prior to analysis. Further, in mixed samples, if there are rare highly-damaged cells, their presence can be obscured by the numerous cells that harbor low levels of DNA lesions. Knowledge about how a minority population of cells responds to DNA damage can have important implications, as it only takes one highly damaged cell to initiate cancer or many other diseases. Another problem many laboratories encounter is that the handling and/or processing of a sample of cells, or a tissue, can introduce DNA damage. For example, when tissue is disaggregated, the levels of DNA damage increase as a result of the stresses involved in tissue disaggregation.
One approach used for assessing the extent of DNA repair is the use of unscheduled DNA synthesis. However, this approach is not useful for assessing directly-induced DNA damage, such as double and single strand breaks.
Structural and numerical chromosome aberrations, as well as sister chromatid exchanges, are alternative ways of detecting damage in DNA, but they can only analyze cells that are in metaphase. Accordingly, some cell types can almost never be analyzed because they rarely are in metaphase, while for all cell types enough cells in metaphase must be gathered while cells in any other phase must be discarded. Further, with respect to structural and numerical chromosome aberrations, information linking the observed aberration back to the cell in which it occurred is nearly impossible to gain because once a metaphase spread is made, cell type information is generally lost. The process is also very slow and labor-intensive, generally requiring extensive microscope time and skilled technicians. It is also not feasible to detect subtle effects of DNA repair deficits or exposures using the aberration method unless sufficient time has elapsed for accumulation of rare aberrations, which in many cases is too late for appropriate intervention to occur. With respect to the sister chromatid exchanges, they are highly transient in nature, so the timing relative to a potential exposure is critical, which in turn makes “false negative” readings a common occurrence.
Micronucleus assays are used to detect DNA fragmentation. While micronucleus assays can be useful for studies of one organ, such as bone marrow, they are of limited value for many other cell types. Further, the assays cannot be used to assess DNA base lesions.
Prior to the present disclosure, three of the more promising methods for DNA damage detection were mass spectrometry, immunohistochemical detection of phosphorylated H2AX, and the comet assay. Mass spectrometry can be useful for precision lesion identification, but at present this approach is not readily amenable to large scale population studies due to the number of cells needed per assay, the technical difficulty of performing the assay, and the cost of the equipment required for analysis. Currently it takes about two weeks to process a set of approximately ten samples. Even under optimal conditions, this approach requires technical expertise and access to a mass spectrometer.
While immunohistochemical detection of phosphorylated H2AX is a sensitive way for measuring DNA double strand breaks, it is technically very difficult to use this assay to assess DNA damage levels in S phase cells. (MacPhail et al. 2003; Han et al. 2006.) It is really only optimal for cells in the G0 or G1 phases, and because many environmental exposures that cause genomic instability do so by interfering with DNA replication during the S phase, this is problematic. This method is further limited by the fact that it only detects double strand breaks. While these are clearly very important lesions, in the case of ultraviolet and aflatoxin exposure, which are two of the best-characterized environmental mutagens, the vast majority of the DNA lesions created by these exposures are base-modifications, not double strand breaks. (Friedberg et al. 2006.)
The comet assay is also a sensitive assay for measuring both the levels of DNA damage and the rate of DNA repair. In a comet assay, cells are generally embedded in agarose, and after electrophoresis is performed on the assay, undamaged DNA generally remains supercoiled and highly compact while damaged DNA more readily migrates during electrophoresis and gives rise to the appearance of a bright nucleoid with a comet-like tail. Unfortunately, using standard methods and/or devices, it takes hours to prepare and process just a single sample. It is a very labor and time-intensive process. For example, completing the incubation steps alone can take approximately four hours. This is due, at least in part, to the fact that current comet assays are performed at room temperature. Further, the feasibility of testing multiple conditions in parallel and/or processing a number of independent samples is severely limited by the current methods and devices. This is due, at least in part, to the potential of overlapping tails of cells being tested. In terms of the area required for a routine comet assay, the density of the cells is primarily limited by interference between cells. Another problem with current comet assays is that there is a lack of standardization that has lead to undesirable variability not only from laboratory-to-laboratory, but from user-to-user from the same laboratory and from slide-to-slide and assay-to-assay from the same user.
At present, clinical assays are not generally available to medical doctors to assess a patient's DNA repair capacity. This information would be invaluable to a person who might be able to avoid cancer simply by avoiding certain exposures. Furthermore, a person's DNA repair information could be used to guide appropriate intervals for cancer screening, such as for early detection of cancers, and even could be used to guide appropriate choices of treatments, for example by preventing chemotherapy-induced lethality in a repair-deficient patient. Aside from cancer, knowledge of a person's DNA repair capacity could guide appropriate selection of other pharmaceuticals to avoid drugs to which a person may be acutely sensitive. Having an assay that directly measures an endpoint that is predictive of how a person would respond to environmental risk factors would also be extremely valuable, which in turn would allow a person to avoid risky behaviors and allow physicians to more accurately weigh the cost-benefit of anti-inflammatory interventions, such as non-steroidal anti-inflammatory drugs. It would be an invaluable tool for both revealing and controlling environmental risk factors. Further, in the research setting, a high-throughput DNA repair assay would be useful for the Gene-Environment Initiative because it could be used to identify as-yet-unknown genetic risk factors that cause a deficiency in DNA repair and thus sensitize particular individuals to certain environmental exposures.
Accordingly, there exists a need for a sensitive, efficient, consistent, and reliable method for analyzing nucleic acid damage and repair in all cell cycle phases. Likewise, there exists a need for devices and systems capable of carrying out a sensitive, efficient, consistent, and reliable analysis of nucleic acid damage and repair in all cell cycle phases. Additional benefits could be realized if such methods, devices, and systems allowed for parallel processing to permit simultaneous analysis of a multitude of samples or conditions.